The objective of this work is to determine how a Mycobacterial secretion apparatus called ESX-1 plays critical roles in important, but apparently disparate, molecular processes: conjugal DNA transfer and virulence. This laboratory has established that ESX-1 secretion and DNA transfer are functionally and genetically intertwined;ESX-1 mutants are defective in DNA transfer. Our studies have allowed us to propose a model in which Mycobacteria customize their ESX-1 secretion profile in response to different physiological and environmental cues. This explains the functional versatility of the conserved ESX-1 secretory apparatus in promoting DNA transfer and survival in a macrophage. The proposed studies will elucidate the impact of these two processes on the biology and pathogenesis of Mycobacteria. Conjugation requires cell-cell contact and involves the unidirectional transfer of DNA from a donor cell to a recipient cell, often resulting in the transfer of large segments of chromosomal DNA. DNA transfer between strains of Mycobacterium smegmatis occurs by a novel mechanism distinct from the prototypical, plasmid-mediated conjugation systems. A long-term goal of the work is to understand the contributions of conjugation to mycobacterial evolution, including the spread of drug resistance and virulence determinants. The ESX-1 secretory apparatus is essential for virulence of Mycobacterium tuberculosis. There is no genetic assay for M. tuberculosis ESX-1 activity, and both ESX-1 substrates and the mechanism of secretion are poorly defined. This proposal will address these gaps by taking advantage of a unique observation that we have made: ESX-1 is essential for DNA transfer in the M. smegmatis recipient strain. The requirement for an intact ESX-1 apparatus for DNA transfer in M. smegmatis provides the first genetic screen for the isolation of ESX-1 mutants. The goals of this proposal are to characterize the process of DNA transfer, to exploit DNA transfer as a genetic tool, to elucidate the mechanism of ESX-1 secretion and to determine the roles of the proteins secreted by ESX-1 in both regulation of DNA transfer and M. tuberculosis virulence. These goals will be achieved using a combination of molecular, genetic and biochemical assays. The specific aims are to: 1. Determine the genetic requirements for conjugal DNA transfer in donor and recipient strains of M. smegmatis in order to characterize this novel mechanism of genetic exchange. 2. Utilize DNA transfer to perform a genome-wide genetic screen to identify genes required for ESX-1 activity, and determine their role in both DNA transfer and virulence. Together, these aims describe an innovative approach to dissect the mechanism of ESX-1 secretion. Moreover, the identification of the secreted proteins, and their extracellular targets will provide significant new insights into their roles in both lateral DNA transfer and M. tuberculosis virulence.